1. Field of the Invention
This invention relates to a gene containing a DNA coding for S-hydroxynitrile lyase, a recombinant vector plasmid gene possessing the gene, and Escherichia coli containing the recombinant vector plasmid gene. This invention further relates to a method for the production of S-hydroxynitrile lyase by the use of recombinant Escherichia coli having integrated the gene mentioned above.
2. Description of the Related Art
The S-hydroxynitrile lyase (EC 4.1.2.37) originating in cassava (Manihot esculenta Crantz) is an effective enzyme for the synthesis of an optically active S-cyanohydrin from an aromatic or aliphatic carbonyl compound and hydrogen cyanide. The synthesis of the optically active cyanohydrin by the use of this enzyme is very useful for the sake of synthesizing various optically active intermediates. Since this enzyme is contained only in the amount of a mere trace in the tissue of cassava (Manihot esculenta Crantz), rose-rubber tree (Hevea brasiliensis), etc., however, it has been difficult to utilize this enzyme on a commercial scale.
An example of the production of this enzyme implemented by the culture of recombinant Escherichia coli integrating therein a gene coding for S-hydroxynitrile lyase originating in cassava (Manihot esculenta Crantz) with a view to solving the problem has been heretofore known (Biotechnol. Bioeng. 53, 332–338, 1997). Since this method obtains an HNL protein by isolating a fusion protein formed in Escherichia coli by using a vector having a malE gene and an HNL gene bonded with a polylinker and subsequently severing the fusion protein at the Factor Xa site of the polylinker, this HNL protein turns out to be a recombinant protein having several amino acids added to the N terminal of the inherent S-hydroxynitrile lyase originating in cassava (Manihot esculenta Crantz), fails to form the folding of protein successfully, and eventually exhibits a quality different from the inherent quality of the protein. Further, since it has the gene originating in a eucaryote integrated into the Escherichia coli which is a procaryote, the recombinant protein consequently obtained has no fully satisfactory specific activity.
An example of the production of the S-hydroxynitrile lyase originating in rose-rubber tree (Hevea brasiliensis) effected by similar gene recombination using a yeast (genus Saceharomyces or genus Pichia), a eukaryotic cell, as a recipient has been known heretofore (official gazette of International Unexamined Patent Publication No. 11-508775). However, this method requires long cultivation time. Also, it is difficult to isolate recombinant proteins from yeast cells whose cell walls are hard to break.
Similarly, an example of the production of the S-hydroxynitrile lyase originating in cassava (Manihot esculenta Crantz) effected by the recombination owing to the use of a yeast episome type expression vector, with the yeast (genus Saccharomyces-or genus Pichia) as a recipient has been known heretofore (official gazette of JP-A-2000-189159 and JP-A-2000-189160). This method similarly suffers from deficiency in specific activity.
In the existing circumstances, the desirability of developing an efficient method for the production of the S-hydroxynitrile lyase by the gene recombination using Escherichia coli has come to find growing recognition in consideration of the fact that this method permits easy high density culture and consequently enables the gene and the protein aimed at to be produced in large quantities, the fact that the cell has a thin wall as compared with a yeast and the fracture of this cell allows easy acquisition of a recombinant protein, and the fact that the method for purifying a target gene and protein has been well established.
Even if the amount of the S-hydroxynitrile lyase to be expressed is simply increased, more often than not the bulk of an inactive body of foreign protein, namely the so-called inclusion body, is formed because them is folding of the foreign protein as by the molecular chaperone does not normally function when the foreign protein is produced in a large quantity in the recipient such as Escherichia coli. The inclusion body results from the alteration of the three-dimensional structure of a target product and manifests its effect on the intrinsic activity of that target product. The necessity of performing a refolding treatment, therefore, arises. Thus, the desirability of developing a method which does not form such an inclusion body as this has been finding popular recognition.